Validating a Xylose Regulator to Increase Polyhydroxybutyrate Production for Utilizing Mixed Sugars from Lignocellulosic Biomass Using Escherichia coli

Polyhydroxybutyrate (PHB) production from lignocellulosic biomass is economically beneficial. Because lignocellulosic biomass is a mixture rich in glucose and xylose, Escherichia coli, which prefers glucose, needs to overcome glucose repression for efficient biosugar use. To avoid glucose repression, here, we overexpressed a xylose regulator (xylR) in an E. coli strain expressing bktB, phaB, and phaC from Cupriavidus necator and evaluated the effect of xylR on PHB production. XylR overexpression increased xylose consumption from 0% to 46.53% and produced 4.45-fold more PHB than the control strain without xylR in a 1% sugar mixture of glucose and xylose (1:1). When the xylR-overexpressed strain was applied to sugars from lignocellulosic biomass, cell growth and PHB production of the strain showed a 4.7-fold increase from the control strain, yielding 2.58 ± 0.02 g/l PHB and 4.43 ± 0.28 g/l dry cell weight in a 1% hydrolysate mixture. XylR overexpression increased the expression of xylose operon genes by up to 1.7-fold. Moreover, the effect of xylR was substantially different in various E. coli strains. Overall, the results showed the effect of xylR overexpression on PHB production in a non-native PHB producer and the possible application of xylR for xylose utilization in E. coli.


Introduction
As a bio-based, bio-degradable polymer, polyhydroxybutyrate (PHB) is a promising alternative to petrochemicalbased plastics [1].PHB has potential applications in many areas, including the medical materials and packaging industry, which therefore necessitates reducing production costs to expand the coverage and sustainability of PHB bioprocessing [2][3][4].One strategy for reducing PHB production cost was to lower the price of raw materials by utilizing lignocellulosic biomass as a carbon source for PHB-producing strains [5].Lignocellulosic biomass is a highly abundant, inexpensive by-product of the agriculture and wood industry, and does not affect the human food industry, which makes it a promising substrate for PHB production [6].However, lignocellulosic biomass becomes a sugar mixture rich in glucose and xylose when pretreated and hydrolyzed; therefore, it is necessary to overcome carbon catabolite repression (CCR) to utilize it as a carbon source for PHB-producing strains [7].For Escherichia coli, glucose is preferred as a carbon source over xylose.As a result, the bacteria only utilize glucose until it is completely depleted, while xylose remains unused [8].This phenomenon occurs because when glucose enters the cell of E. coli, dephosphorylated phosphotransferase protein (EIIA) is produced in the phosphotransferase system, which then acts to exclude and prevent utilization of secondary metabolic sugars.Dephosphorylated EIIA enzyme binds to an allosteric site of the promoter and prevents formation of cAMP, which is necessary for utilizing other sugars [9,10].
As the xylose operon in bacteria has xylAB (xylose metabolism), xylFGH (xylose transporter), and xylR (xylose regulator) [11], many researchers have applied genetic engineering and overexpressed these genes to facilitate xylose utilization.Moreover, xylR in particular is one of the most commonly used genes for growing E. coli in xylose or mixed sugars, while overexpressed xylR or introduction of an xylR mutant to promote xylose consumption or simultaneous consumption of mixed sugars have been reported to have a positive effect on xylose consumption [12][13][14][15].Therefore, bacterial growth could be enhanced with xylose or mixed sugars, such as glucose/xylose or xylose/arabinose, to some extent.Previous studies have already shown that the latency at transition from glucose to xylose is controlled by intracellular xylR availability [15].In addition, it was reported that xylR overexpression in E. coli enabled its exponential growth in xylose and increased production of biochemicals, such as isobutanol and ethanol, in glucose/xylose mixture sugars [16,17].Burkholderia sacchari, which is an industrially viable strain for the production of xylitol, xylonic acid, and PHB, was engineered with xylose-related genes that resulted in a considerable increase in growth and PHB production via xylR overexpression [13].Interestingly, despite these efforts, studies on PHB production via xylR overexpression in E. coli have not been conducted yet.As PHB is accumulated inside the cells, the impact of mixed sugar seems to be different from that of secreted products such as ethanol and isobutanol in E. coli, while a limitation of space required to produce PHB in a non-native PHB producer would have a different effect from that seen in native PHB-producing bacteria.In addition, considering that the cost of PHB production is highly dependent on the cost of substrate, inexpensive and abundant starting materials, such as lignocellulosic biomass, could improve the overall economy of PHB production, and feasibility testing of PHB production in E. coli with overexpressed xylR could be very helpful in further applications.Although the pretreatment cost of lignocellulosic biomass is still an obstacle, as part of the biorefinery process that breaks down biomass into several valuable products and makes the most of them, improving PHB production by increasing consumption of xylose could contribute to improving the overall economic efficiency of the process and reducing surplus resources.To confirm this, we examined the effect of xylR on PHB production in E. coli by introducing a PHB production vector, pLW487 [18][19][20], into E. coli and overexpressing xylR.

Bacterial Strains and Culture Condition
The bacteria and plasmids used in this study are listed in Table 1.E. coli DH5α was used for gene cloning for PHB production and xylose metabolism.The plasmid was extracted using Exprep Plasmid SV Kit (GeneAll, Korea).In addition, the extracted plasmid was inserted into various E. coli expression competent cells prepared in advance using the heat shock method.The cell culture was carried out in 5 ml of lysogeny broth (Difco laboratories, USA), and spectinomycin and kanamycin were selectively used for pLW487 and pRSFDuet-1.For PHA production, E. coli strains were cultured in 5 ml M9 minimal medium containing a 1% (w/v) glucose and xylose mixture, 0.1% (w/v) yeast extract, and 0.1 mM (w/v) isopropyl β-d-1-thiogalactopyranoside (IPTG) for 72 h at 30°C.

RT-qPCR Analysis
For comparing the transcription level of genes related to xylose metabolism, E. coli HJ010 and HJ020 were cultured in 5 ml M9 minimal media containing 1% (w/v) sugar mixture (0.5% glucose + 0.5% xylose), 0.1% (w/v) yeast extract, and 0.1mM (w/v) IPTG for 72 h at 30°C.The mRNA was extracted using the RNeasy Mini Kit (Qiagen, Germany) as set forth in the manual.For cDNA synthesis, the SuperScript III First-Strand Synthesis System (Invitrogen, USA) was used.After equalizing the concentration of the synthesized cDNA equally, Realtime PCR was performed with StepOnePlus (Applied Biosystems, USA) using TOPreal qPCR 2X PreMIX (SYBR Green with high ROX, Enzynomics, Korea).

Analysis of Dry Cell Weight and PHB Production
PHB production was determined using gas chromatography and other methods as described in previous studies, with slight modifications [21][22][23].For analysis, culture samples were centrifuged at 10,000 ×g for 20 min, washed with deionized water twice, and suspended in 1 ml water.The suspended sample was transferred to Δpflb, ΔldhA, ΔadhE, Δfnr, containing pACYC::pntAB, BW25113(DE3) derivative [29] JM109(DE3) recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, λ-, Δ(lac-proAB), [F' traD36, proAB, lacI q laqZΔ M15] This study Plasmids pLW487 Spec R pEP2-based, plasmid carrying bktB, phaB and phaC under trc promoter from R.eutropha Km R , pRSFDuet-1-based plasmid carrying xylR from Escherichia coli under T7 promoter [16] weighted Teflon-stopped glass vials, and after lyophilization, the weight of the glass vials was re-measured to confirm the dry cell weight (DCW).For methanolysis of PHB samples, a mixture of 1 ml chloroform and 1 ml of methanol/sulfuric acid (85:15 v/v) was added to the vials and then incubated at 100°C for 2 h.The samples were then cooled in a refrigerator for 20 min to prevent the evaporation of chloroform.After adding 1 ml water, samples were mixed twice by vortexing, 10 s apart.The organic phase (bottom) was extracted using a pipette and then moved to a tube containing anhydrous sodium sulfate (Na 2 SO 4 ) to remove residual moisture.Filtered samples were then injected into a GC system (Young Lin Tech., Korea) using a DB-Wax column (30 m × 0.32 mm × 0.5 μm; Agilent Technologies, USA).The split ratio was 1:10.Helium was used as the carrier gas, with a flow rate of 3.0 ml/min.A 2 μl portion of the organic phase was injected using an autosampler.The inlet was maintained at 210°C.The column oven was held at 80°C for 5 min, heated to 220°C at 20°C/min, and then held at 220°C for 5 min.Peak detection was performed using a flame ionization detector, which was maintained at 230°C.

Lignocellulosic Biomass and Residual Sugar Analysis
The lignocellulosic biomass hydrolysate used in this study is a saccharomate® derived from Miscanthus and pine tree purchased from Sugaren Co., Ltd.(Korea).
For analysis of glucose and xylose, culture samples were centrifuged at 10,000 ×g for 10 min, and 1 ml of supernatant was filtered using a 13 mm syringe filter with 0.22 μm PVDF membrane (Chromdisc, Korea).
High-performance liquid chromatography on a PerkinElmer system equipped with a refractive index detector (Waltham, USA) and an Aminex HPX-87H column (300 × 7.8 mm internal diameter) was used to analyze the concentration of various carbon sources.A mobile phase of 0.008 N H 2 SO 4 at a flow rate of 0.6 ml/min was used and the oven temperature was constantly maintained at 60°C during the operation.

SDS-PAGE for Comparing the Amount of Xylose Regulator
To compare XylR amounts between various E. coli strains, six E. coli strains were cultured under optimized conditions (Fig. 2) in this experiment.Thereafter, 1 ml of the culture was divided, and the number of cells was adjusted equally to ensure that the optical density was 7.0-8.0 at 600 nm.Cell pellets were obtained via centrifugation at 15,628 ×g for 5 min and protein was extracted using Bugbuster.It was then mixed with 5X SDSloading buffer and heated at 99°C for 5 min using a PCR machine (HushRun™, Biofact, Korea).The sample was loaded into the pre-prepared gel and SDS-PAGE was performed at 120 V for 2 h.

Transmission Electron Microscopy
To prefix the cell specimens, 1 ml of each culture sample was collected, centrifuged, and mixed with Karnovsky's fixative solution containing 2% formaldehyde.The samples were then fixed with 1% osmium tetroxide in 0.05 M sodium cacodylate buffer and dehydrated in ethanol gradually (v/v) (30, 50, 70, 80, 90, and 100%).The transition step was performed using propylene.The samples were settled in different ratios of propylene oxide and Spurr's resin (v/v) (1:1 and 1:2); 100% Spurr's resin at 70 o C in a dry oven was used to solidify the samples for ultramicrotome sectioning.The specimens were placed on a grid and observed under a transmission electron microscopy (TEM), with an accelerating voltage of 80 kV.TEM images of the cells and PHB granules were obtained at ×15k, ×25k, and ×40k magnification.

PHB Production at Different Glucose and Xylose Ratios
As xylR is one of the most commonly used genes to overcome carbon catabolite repression (CCR), we aimed to validate the effectiveness of xylR on the production of PHB in mixed sugars using E. coli.To achieve this goal, we constructed HJ020 strain by introducing the PHB production vector pLW487 and the xylR overexpression vector pHJ02 in E. coli BL21 (DE3) strain.The pLW487 vector has bktB, phaC, and phaB genes inserted instead of the phaCAB operon of Curpriavidus necator, considering its extensibility to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) as well as polyhydroxybutyrate (PHB).E. coli HJ010 strain with pLW487 and pRSFDuet-1 was used as the control strain.To see the effect of xylR overexpression in glucose and xylose single and mixed sugars contained in lignocellulosic biomass, the DCW and PHB accumulation of HJ010 and HJ020 were confirmed at various ratios of glucose and xylose in M9 minimal media with 0.1% (w/v) yeast extract.When glucose was used as a single carbon source, the DCW (2.42 g/l) and PHB (1.11 ± 0.01 g/l) of HJ020 were greater than the DCW (1.39 ± 0.07 g/l) and PHB (0.28 ± 0.01 g/l) of the control strain (Fig. 1).When xylose was used as a single carbon source, it was confirmed that the DCW of HJ020 strain was 1.91 ± 0.05 g/l and PHB yield was 0.98 ± 0.04 g/l, which increased compared to that seen in the control strain: DCW, 1.51 ± 0.03 g/l, and PHB, 0.40 g/l.
In contrast, the highest PHB accumulation (1.14 ± 0.09 g/l) was achieved for HJ020 when incubated in a 1:1 mixture of glucose and xylose, with a DCW of 2.11 ± 0.11 g/l.The higher the ratio of xylose at 1% of the total carbon source concentration, the lower the cell growth and PHB accumulation in both control strain and HJ020, suggesting that E. coli has a higher preference for glucose, even if xylR is overexpressed.However, the reasons for high growth and PHB accumulation in the 1:1 ratio of glucose and xylose are unclear.We could only interpret this as a result of the increased xylose consumption having a positive effect, since we could see that the xylose consumption was clearly increased (Fig. 3D).PHB production in E. coli requires acetyl-CoA as a precursor and NADPH as a coenzyme.Therefore, for the PHB accumulation of E. coli, the use of glucose as a pentose phosphate pathway is recommended rather than glycolysis.In addition, since xylose can expand the pool of the nonoxidative pentose phosphate pathway, xylose has an advantage in that it can increase the consumption of glucose to the oxidative pentose phosphate pathway.We conducted the following experiment on a 1:1 glucose-xylose mixture with the highest PHB accumulation.

Optimization of Sugar, Nitrogen and IPTG Concentration
E. coli HJ020 has been genetically engineered to allow PHB accumulation, minimizing catabolite repression in lignocellulosic biomass, which primarily contains glucose and xylose.We optimized the concentration of carbon sources, nitrogen sources and IPTG to maximize PHB accumulation before introducing HJ020 strain into the lignocellulosic biomass.
First, DCW and PHB accumulation of HJ020 were confirmed at concentrations of 1-6% of glucose and xylose mixture (1:1).At 1% sugar mixture, the DCW of HJ020 was 1.82 g/l, and PHB yield (1.33 g/l) of 73.22% of the total cell weight was obtained.There was no marked change in cell growth and PHB production even though the concentration of carbon sources increased (2~6%).As shown in the Fig. 2B, E. coli HJ020 strain did not consume more sugar even with an increased concentration of carbon.For optimization of the nitrogen source concentration, the cell growth and PHB production of E. coli HJ020 were confirmed at a concentration of 0.1 to 0.3% of yeast extract, a nitrogen source primarily used in E. coli culture.The optimal yeast extract concentration was 0.1%, at which time HJ020 accumulated 53.88% of PHB (1.14 ± 0.09 g/l) relative to the total cell weight (2.11 ± 0.11 g/l).IPTG concentration was optimized at 1% sugar mixture and 0.1% yeast extract to control the optimal expression of xylR expressed under the T7 promoter.As shown in the Fig. 2D, it was confirmed that HJ020 produced the highest DCW (1.21 ± 0.01 g/l) and PHB (0.50 ± 0.02 g/l) at 0.1mM IPTG concentration.

Time-Dependent Monitoring of PHB Production and Sugar Utilization
Under optimized conditions, time-dependent cell growth, PHB accumulation, and sugar mixture consumption of E. coli HJ010 and HJ020 were confirmed.For HJ010 (control), the highest DCW (1.2 ± 0.1 g/l) was obtained by quickly consuming only glucose for 24 h after incubation initiation.In addition, all remaining glucose was consumed between 24 and 48 h, and the highest PHB production (0.25 ± 0.00 g/l) was confirmed at 48 h (Fig. 3).Considering that E. coli DH5a and BL21 (DE3) strains consumed all glucose within 24 h under the same culture conditions (Fig. S1), it is possible that the accumulation of PHB in cells due to the introduction of vector reduced the rate of glucose consumption in E. coli.On the other hand, in the case of xylR-overexpressing E. coli HJ020 strain, the highest DCW (2.0 ± 0.1 g/l) and PHB (1.09 g/l) production were confirmed at 48 h, and in the case of PHB, it was 4.45-fold higher than HJ010 (Figs. 3A and 3B).As a result of analyzing the residual sugar consumption in HJ020, we interpret the enhanced cell growth and increased PHB production as a direct outcome of the efficient utilization of xylose attributed to the overexpression of xylR.In contrast to HJ010 strain, which did not consume xylose, HJ020 strain was shown to have consumed 46.53 ± 0.58% of xylose out of 5 g/l of xylose (Fig. 3D).Because of xylR overexpression, the consumption rate of glucose decreased slightly compared to control.The decrease in the consumption rate of glucose due to this xylR overexpression is similar to previous studies in which xylR mutant introduction increased xylose consumption and reduced glucose consumption [17].As a result, the overexpression of xylR could have a positive effect on the PHB accumulation of E. coli, as the rate of consumption of glucose decreased slightly, although the total consumption of mixed sugars increased at 72 h.Further, we also confirmed the morphology of HJ010 and HJ020 at 72 h by using transmission electron microscopy (TEM) (Fig. 4).As a result,

Fig. 3. Time-dependent (A) dry cell weight (DCW), (B) PHB production and sugar utilization of (C) E. coli HJ010 (control) and (D) E. coli HJ020 (xylR) in optimized condition.
HJ020 strain overexpressed xylR accumulated more PHB in the cell and generally showed a larger cell size than the control strain.

PHB Production with xylR-Overexpressing Strain by Using Lignocellulosic Biomass Hydrolysate
To confirm the effect of xylR overexpression in the actual lignocellulosic biomass, PHB production and cell growth were confirmed from lignocellulosic biomass hydrolysate using HJ020.Miscanthus-derived biosugar contained a high proportion of glucose showing 113:4 ratio to xylose, and pine tree-derived biosugar contained a high proportion of xylose, showing a 36:103 ratio for glucose to xylose (Table S1).As shown in Fig. 5, HJ020 showed lesser PHB production than control with hydrolysate of Miscanthus because of the extremely high ratio of glucose to xylose.In the case of pine tree, HJ020 accumulated 4-times more PHB (1.57± 0.11 g/l with 50.08 ± 4.97% of PHB content) than control at 1% pine tree biosugar, and there was no significant difference in cell growth.The maximum PHB concentration and cell growth were achieved in the Miscanthus + pine tree sugar mixture, with a 74:53 ratio of glucose to xylose.HJ020 increased PHB yield (2.58 ± 0.02 g/l with 58.44 ± 3.21% of PHB content) by 4.7 times compared to that seen in the control, and the DCW (4.43 ± 0.28 g/l) further increased by 1.6  times.Although xylR overexpression was effective in high-xylose conditions, it seemed to be the best with a similar ratio of glucose and xylose.On the other hand, cell growth and PHB production when using lignocellulosic biomass hydrolysate were generally higher than when using glucose and xylose.This was contrary to our expectation that cell growth would decrease because the lignocellulosic biomass hydrolysate contains inhibitors, such as furfural and acetic acid.The lignocellulosic hydrolysate contains a variety of amino acids, peptides, metal ions, and aromatics, as well as carbohydrates, which were expected to accumulate high DCW and PHB by supplementing trace elements lacking only with the use of yeast extract [24][25][26][27].

Analysis of Xylose Operon Transcription Level with xylR Introduction
To evaluate the transcription level of xylR and the resulting expression of xylose operon, E. coli HJ020 strain was cultured in M9 minimal medium with 1% sugar mixture for 72 h, and then mRNA was extracted and RT-qPCR was performed.E. coli HJ010 strain was used as control.The overexpressed xylR under the T7 promoter was 205.6fold more expressed than the control.In HJ020, a strain overexpressing xylR, expression of xylA (1.(DE3), HR200 (DE3), and KSYH (DE3)) were introduced with pLW487 and pHJ02 (xylR).The culture was conducted under optimized conditions in the previous experiment, and PHB and DCW were measured before and after xylR introduction.
As expected, BL21 (DE3) showed increased PHB production by overexpression of xylR.In the case of E. coli C41 (DE3), there were no significant differences observed in terms of DCW when xylR was overexpressed.PHB production was increased by 4 times in BL21 and 1.6 times in C41, accumulating PHB of 0.86 g/l and 0.96 ± 0.03 g/ l, respectively (Fig. 7A).This suggested that xylR overexpression in E. coli had a positive effect on PHB production with the mixed sugar.However, overexpression of xylR did not have a positive effect on all E. coli strains.When xylR was overexpressed in C43 (DE3), JM109 (DE3), KSYH (DE3), and HR200 (DE3) strains, both the DCW and PHB accumulation were shown to be decreased.This type of a result was reported in Burkholderia sacchari.When xylR expression was induced with low-level IPTG (6.25 μM), a 66% increase in cell growth was achieved, and highlevel IPTG induction (50 or 500 μM) was reported to delay growth [13].In addition, in the case of E. coli MG1655 strain, it has been reported that high expression levels of xylR in arabinose and xylose mixed sugars caused growth delay [12].As stated above, the high expression level of xylR may have negatively affected growth, and we hypothesized that the four strains with reduced growth would have expressed higher amounts of xylR at the same IPTG concentration.To prove this hypothesis, SDS-PAGE and RT-PCR were attempted to compare XylR protein amounts and xylR expression levels for various E. coli strains (Figs.7B and 7C).However, there was no significant correlation between the experimental results and the decline in growth in E. coli strains.Even the comparison between BL21 (DE3) and C41 (DE3), which showed increased PHB production, the correlation was hard to detect.Moreover, strains with decreased PHB production showed quite different patterns of protein and mRNA expression.As a result of confirming the expression of xylR through RT-PCR and SDS-PAGE, the expressions of mRNA and protein between strains were not proportional.In the case of C41, C43, and KSYH strains, mRNA expression was high, but protein expression was relatively low.On the other hand, in the case of JM109 and HR200, the expression of mRNA was relatively low, but the expression of protein was high.The effect of xylR in one strain can be associated with the expression of xylR, but the effect of xylR on multiple strains can be difficult to relate to the expression.This relationship between xylR overexpression and decrease in growth may have more complex causes than reported and expected.Considering various E. coli strains had many manipulations with sugar metabolisms, the interaction of sugar metabolisms seemed to affect xylose utilization and PHB production combined with xylR.color denotes Escherichia coli expression strains with pLW487 for PHB production.

Conclusion
We found that E. coli HJ020, an xylR-overexpression strain with pLW487 inserted for PHB production, is effective in PHB production in a 1% mixture of glucose and xylose (1:1).Under optimized conditions, the DCW (2. 0 ± 0.1) of HJ020 was 1.7-fold higher than the control strain and produced 4.45-fold more PHB (1.09 ± 0.00).Furthermore, the xylR overexpression of HJ020 was effective in lignocellulosic biomass hydrolysate rich in glucose and xylose.This strain produced 2.58 ± 0.02 g/l of PHB in a 1% Miscanthus and pine tree mixture and accumulated 4.7-fold more PHB than the control strain.We confirmed that these results were due to the effective activation of xylose operon by overexpressed xylR.xylR overexpression can be presented as a strategy to increase PHB production using E. coli BL21 from lignocellulosic biomass.
To verify the universality of xylR overexpression, we tested its effects on the production of PHB in different E. coli expression strains.However, among the six strains, only BL21 and C41 strains showed an increase in PHB production under xylR overexpression, and in the remaining four strains (C43, JM109, HR200 and KSYH), xylR overexpression resulted in a decrease in DCW and PHB production.Although the positive effects of xylR in BL21 are apparent, the control of CCR might be more complex and strain-dependent than what has been explored in this study.

Fig. 6 .
Fig. 6.The mRNA expression of xylAB and xylFGH operons because of xylR overexpression was observed using RT-qPCR.

Fig. 5 .
Fig. 5. Comparison of PHB production with and without xylR.Hydrolysate derived from Miscanthus was used as glucose, and hydrolysate derived from pine tree was used as xylose.colordenotes Escherichia coli HJ010.colordenotes E. coli HJ020.

Fig. 7 .
Fig. 7. Changes in (A) DCW and PHB obtained via xylR overexpression in various E. coli expression strains.
color denotes E. coli expression strains with pLW487 and pHJ02 for xylR overexpression.(B) SDS-PAGE for the comparison of amount of XylR in different E. coli strains and (C) comparison of xylR expression level via RT-PCR in various E. coli expression strains were performed.